L-Glutamate (L-Glu) is recognized as the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Current estimates suggest that about 30-40% of the synapses in the mammalian CNS are glutaminergic. Glutaminergic neurons are localized in nearly all regions of the brain, with particularly high densities in the areas involved in learning and memory, such as cerebral cortex and hippocampus. In the mammalian CNS, the excitatory amino acid transporter (EAAT) family of proteins is responsible for the high-affinity sodium-dependent uptake of L-Glu into both astroglial cells and neurons. Among the five subtypes of EAAT receptors, EAAT-1 and EAAT-2 are the major contributors to L-Glu uptake from the synapse.
If L-Glu levels are not properly regulated by the EAATs and become excessive, it can result in the over-activation of excitatory amino acid (EAA) receptors and induce neuronal pathology in a process known as excitotoxicity. Rapid increases in intracellular Ca2+ that follow the excessive activation of NMDA clas of EAA receptors can trigger both necrotic and apoptotic pathways and ultimately result in cell death. Energy consumption and increased rates of reactive oxygen species (ROS) generation are also associated with the over-activation of glutamate receptors and mitochondrial damage. Excitotoxicity is now well-recognized as a primary or secondary mechanism underlying the pathology observed in growing number of acute insults to the CNS such as ischemia, hypoglycemia, spinal cord injury and traumatic brain injury. Excitotoxicity also contributes to the development or progression of chronic neurological and neurophysiological disorders such as amyotrophic lateral sclerosis (ALS), epilepsy, schizophrenia, Huntington's disease, and Alzheimer's disease.
For example, neuronal cell death in ALS is the result of over-activation of neuronal cells due to excess extracellular L-Glu. In a normal spinal cord and brain stem, the level of extracellular L-Glu is maintained at low micromolar levels in the extracellular fluid because glial cells, which function in part to support neurons, use EAAT-2 to rapidly sequester L-Glu and restrict its access to EAA receptors. A deficiency in the normal EAAT-2 protein in patients with ALS, was identified as being important in the pathology of the disease (See, e.g., Meyer et al., J. Neurol. Neurosurg. Psych. 65:594-596 (1998); Aoki et al., Ann. Neurol. 43:645-653 (1998); and Bristol et al., Ann Neurol. 39:676-679 (1996)). One explanation for the reduced levels of EAAT-2 is that EAAT-2 is spliced aberrantly (Lin et al., Neuron 20:589-602 (1998)). The aberrant splicing produces a variant with a deletion of 45 to 107 amino acids located in the C-terminal region of the EAAT-2 protein (Meyer et al., Neurosci. Lett. 241:68-70 (1998)). Due to the lack of, or defectiveness of EAAT-2, extracellular L-Glu accumulates, causes neurons to be excessively activated, and includes excitotoxic pathology. Thus, the accumulation of glutamate has a toxic effect on neuronal cells because continual firing of the neurons leads to early cell death.
Small molecule agents that inhibit transport of the L-Glu by antagonizing EAATs in the CNS are considered useful molecular entities that can alter synaptic concentrations of L-Glu, can serve as in vitro and in vivo pharmacological probes of the EAATs, and in their radiolabeled forms can detect EAATs in CNS and peripheral tissues. Several EAAT inhibitors with high inhibitory potency have been described, including inhibitor variants described by Greenfield et al. (Bioorg. Med. Chem. Lett. 15:4985 (2005)). There remains a need, however, for EAAT antagonists that can penetrate the blood-brain barrier (BBB) to apply pharmacologically-effective concentrations to tissues and cells of the CNS.
Finally, as excitotoxic injury promoted by elevated L-Glu can result from altered regional astrocyte EAAT-2 activity or cell surface expression, EAAT antagonists that can be visualized by radiographic imaging, such as positron emission tomography (PET), are needed to allow for the quantitative assessments of these in vivo changes. The chemical synthesis and preparation of complex organic compounds for use as radiographic “tracer” compounds has proven to be unpredictable and difficult in the art (see, e.g., Lee et al., Science 334:639 (2011)).